Drug Metabolism & Disposition - DMD #44271 In Vitro ......2012/03/26 · be predominantly estrone...

DMD #44271

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In Vitro Evaluation of the Interaction Potential of Irosustat with Drug Metabolizing Enzymes.

Verònica Ventura, Josep Solà, Concepción Peraire, Françoise Brée and Rosendo Obach.

Ipsen Pharma, S.A. Pharmacokinetics and Drug Metabolism Department. Sant Feliu de

Llobregat, Barcelona, Spain. (V.V., J.S., C.P., R.O.)

Xenoblis. Parc d’Affaires de la Bretèche. Saint Grégoire, France (F.B.)

DMD Fast Forward. Published on March 26, 2012 as doi:10.1124/dmd.111.044271

Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 26, 2012 as DOI: 10.1124/dmd.111.044271

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Running Title: Metabolic drug-drug interactions of Irosustat

Corresponding Author:

Josep Solà (e-mail: [email protected])

Ipsen Pharma S.A. Crta. Laureà Miró, 395. 08980 Sant Feliu del Llobregat. Barcelona. Spain

Telephone: +34 93 685 81 00 / Fax: +34 93 685 10 53

Number of text pages: 46 (including references and tables)

Tables: 7

Figures: 3

References: 37

Abstract: 250

Introduction: 746

Discussion: 1490

Nonstandard abbreviations used are: AI, aromatase inhibitor; Cmax,ss, maximum plasma

concentration at steady state; CT, threshold cycle; DDI, drug-drug interaction; HLM, human liver

microsomes; HPLC, high performance liquid chromatography; P450, cytochrome P450; PCR,

polymerase chain reaction; RT, reverse transcription; STS, steroid sulfatase; TDI, time-dependent

inhibition; UGT, UDP glucuronosyltransferase;


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Abstract

Irosustat is a first-generation irreversible steroid sulfatase inhibitor currently in development for

hormone-dependent cancer therapy. To predict clinical drug-drug interactions between irosustat

and possible concomitant medications, the inhibition/induction potential of irosustat on the main

drug metabolizing enzymes was investigated in vitro. The interaction of aromatase inhibitors on

the in vitro metabolism of irosustat was also studied. Irosustat inhibited CYP1A2 activity in

human liver microsomes through the formation of its desulfamoylated degradation product and

metabolite 667-coumarin. CYP1A2 inhibition by 667-coumarin was competitive showing a Ki of

0.77 µM, a concentration exceeding only by 5-fold the highest steady state maximum

concentration of 667-coumarin in human plasma at the irosustat recommended dose. In addition,

667-coumarin metabolites enhanced the inhibition of CYP1A2 activity. Consequently, further

clinical interaction studies of irosustat with CYP1A2 substrate drugs are strongly recommended.

667-coumarin also appeared as a competitive inhibitor of CYP2C19 (Ki = 5.8 µM) in human

liver microsomes, and this inhibition increased when assessed in human hepatocytes. Inhibition

of CYP2C19 enzyme activity was not caused by repression of CYP2C19 gene expression.

Therefore, additional mechanistic experiments or follow-up with clinical evaluation are

recommended. Irosustat neither inhibited CYP2A6, 2B6, 2C8, 2C9, 2D6, 2E1, 3A4/5 and UGTs

1A1, 1A4 and 2B7 activities, nor induced CYP1A2, 2C9, 2C19 or 3A4/5 at clinically relevant

concentrations. Finally, results in human liver microsomes indicate that no changes in irosustat

pharmacokinetics are expected in vivo resulting from inhibition of irosustat metabolism in case

of concomitant medication or irosustat-aromatase inhibitor combination therapy with letrozole,

anastrozole or exemestane.


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Introduction

Irosustat, (BN83495, 667 COUMATE, STX64), is a first-generation irreversible steroid

sulfatase (STS) inhibitor for hormone-dependent cancer therapy. STS catalyzes the formation of

estrone and dehydroepiandrosterone from their sulfate conjugates. Both compounds are further

converted by 17β-hydroxysteroid dehydrogenase type 1 to estradiol and androstenediol,

respectively, which are described to promote tumour growth (Reed et al., 2005; Foster, 2008).

STS and aromatase are considered as the key enzymes in the two main pathways of estrogen

synthesis in peripheral tissues of postmenopausal women, where estrogens are formed

exclusively, and in whom breast cancer most frequently occurs. While the aromatase pathway is

already targeted in breast cancer treatment by widely prescribed aromatase inhibitors (AIs) such

as letrozole, anastrozole, and exemestane (Mouridsen et al., 2003; Nabholtz et al., 2003;

Paridaens et al., 2003), increasing amounts of evidence support the role of STS as an important

source of estrogens. STS activity is higher than aromatase activity in normal and malignant breast

tissue (James et al., 1987), and the origin of estradiol in breast cancer tissue has been described to

be predominantly estrone sulfate (Santner et al,. 1984). Besides, STS expression is an important

prognostic factor in human breast carcinoma (Suzuki et al,. 2003). Irosustat inhibits STS activity

in vitro and in vivo in tumour bearing rodents (Woo et al., 2000; Foster et al., 2006), in which

regression of mammary tumours has been also demonstrated (Purohit et al., 2000). Also, irosustat

is the first STS inhibitor tested in phase I clinical trials in postmenopausal women with advanced

metastatic hormone-dependent breast cancer (Stanway et al., 2006).

Irosustat structure is a tricyclic coumarin-based sulfamate, and the presence of the

sulfamoyl-ester group is indispensable for its STS inhibitory activity (Figure 1). Irosustat


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undergoes spontaneous desulfamoylation at physiologic pH (Ireson et al., 2003) and also as a

result of its mechanism of inhibition of STS (Woo et al., 2000), leading to the formation of its

main degradation product and metabolite, 667-coumarin. Data from clinical trials indicate that

the mean steady state maximum plasma concentrations (Cmax, ss) of irosustat and 667-coumarin

are around 0.23 µM (range 0.05-0.41 µM) and 0.08 µM (range 0.02-0.15 µM), respectively,

following 40 mg daily irosustat oral doses (recommended dose). Both compounds are highly

bound to plasma proteins. Irosustat and 667-coumarin plasma free fractions are 1.3 and 1.1%,

respectively (unpublished data).

Previous metabolism studies (Ventura et al., 2011) revealed that irosustat and 667-

coumarin are primarily metabolized towards different hydroxylated derivatives in liver

microsomes from preclinical species and humans. These metabolites and also 667-coumarin are

further conjugated with sulfate and/or glucuronic acid (Figure 1). The human enzymes

responsible of the primary transformation of irosustat are the cytochrome P450 enzymes (P450s):

CYP2C8, CYP2C9, CYP3A4/5, and to a lesser extent, CYP2E1. A minor contribution of

CYP1A2 and CYP2C19 may not be fully excluded. The formation of most of the primary

metabolites can be catalyzed by two or more P450 enzymes, meaning that compensatory

mechanisms in irosustat metabolism are likely to occur in vivo, minimizing the risk of

interactions due to P450 inhibition by co-administered drugs or dietary constituents.

The aim of the present work was to investigate the inhibition/induction potential of

irosustat on drug metabolizing enzymes in order to predict drug-drug interactions (DDIs)

between irosustat and possible concomitant medications in the clinical setting (Pelkonen et al.,

1998; Weaver, 2001). The inhibition experiments were performed on the nine major human drug

metabolizing P450s: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6,


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CYP2E1, and CYP3A4/5, using human liver microsomes (HLM) and P450 probe substrates. The

potential inhibitory effect of irosustat on the three main UDP glucuronosyltransferase (UGT)

enzymes involved in drug metabolism: UGT1A1, UGT1A4, and UGT2B7 (Williams et al., 2004)

was also studied using recombinant enzymes. Finally, the induction potential of irosustat on the

four major inducible human P450s: CYP1A2, CYP2C9, CYP2C19, and CYP3A4/5, was assessed

in human hepatocytes.

Because AIs are potential medications for combined therapy with irosustat (Woo et al.,

2011), the prediction of possible interactions between AIs and irosustat was assessed specifically

for letrozole, anastrozole, and exemestane. From published data, letrozole inhibits CYP2C19

activity (Ki = 42.2 µM) in HLM (Jeong et al., 2009), and anastrozole inhibits CYP2C9, CYP3A

activities (both with a Ki of 10 µM) and CYP1A2 activity (Ki = 8 µM, Grimm and Dyroff, 1997).

Exemestane does not inhibit CYP1A2, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 activies

(Aromasin® prescribing information, 2005), however, no information is available on its CYP2C8

inhibition potential. Therefore, the effect of the three AIs on irosustat metabolism was

investigated in HLM.


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Methods

Chemicals. Test compounds irosustat [purity by high performance liquid chromatography

(HPLC) 99.6%] and 667-coumarin (purity by HPLC 99.1%) were synthesized in Panchim (Evry

Cedex, France). Most reagents including P450-specific substrates, inhibitors, inducers and

reference standards were purchased from Sigma-Aldrich (St. Louis, MO), except where indicated

otherwise. Hydroxybupropion was purchased from BD-Gentest (Woburn, MA). S-mephenytoin

from Tebu-Bio (Le Perray en Yvelines Cedex, France) and Toronto Research Chemicals (North

York, Canada). 7-hydroxy-4-methylcoumarin glucuronide from Alfa Aesar (Ward Hill, MA). AIs

were purchased from different suppliers: letrozole from Toronto Research Chemicals, anastrozole

from Sequoia Research Products (Berkshire, UK), and exemestane from Selleck Chemicals LLC

(Houston, TX). Solvents for HPLC analysis were of analytical or HPLC grade. HLM were

purchased from Biopredic International (Rennes, France) and from Xenotech LLC (pool of 50

mixed gender separate donors, Lenexa, KS). Human freshly isolated hepatocytes from different

women donors were obtained from Biopredic International. Recombinant human UGTs 1A1,

1A4, and 2B7 were all purchased from BD-Gentest.

P450 Enzyme Activity Inhibition. The potential inhibitory effect of irosustat on the

following P450s: CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4/5 was determined in

incubations of HLM with increasing concentrations of irosustat (working solutions in

acetonitrile), and a single concentration of each P450 probe substrate (Table 1). Three substrates

were used to assess CYP3A4/5 inhibition: testosterone, midazolam and nifedipine (Kenworthy et

al., 1999; Galetin et al., 2002). Irosustat concentrations ranged from 0 to 50 µM. All incubations

were performed at 37ºC as indicated in Table 1, in triplicate and using two experimental


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conditions: (a) direct inhibition: simultaneous incubation of HLM with irosustat and P450

substrate; and (b) time-dependent inhibition (TDI): 30 minutes preincubation of HLM with

irosustat in the presence of reduced NADPH (or NADPH-generating system consisting on 4 mM

D-glucose 6-phosphate, 5 mM MgCl2, 2 IU/ml glucose 6-phosphate dehydrogenase and 1 mM β-

NADP) before the addition of the P450 substrate. Reference P450 inhibitors were incubated as

positive controls at a single active concentration (see Table 1). Incubation mixtures without

irosustat and reference inhibitor, without HLM, without substrate and without cofactor were used

as controls (n=1). The solvent composition (Table 1) was set to be constant in all assays, and did

not exceed 2% of the total incubation volume. The inhibitory effect of 667-coumarin (working

solutions made in acetonitrile) on CYP1A2 and CYP2C19 was also investigated under the same

incubation conditions.

Determination of IC50. When more than 50% inhibition was found, IC50 were calculated

by fitting the inhibitory effect sigmoid Emax equation: V=V0·(1-(Cγ/(Cγ+IC50γ))) to the obtained

enzyme activity data by means of least squares non-linear regression, using WinNonlin software

(Pharsight, Mountain View, CA). For CYP1A2, IC50 were calculated by fitting the inhibitory

effect sigmoid Emax equation that considers a residual activity that cannot be inhibited: V=V0-(V0-

V∞)(Cγ/(Cγ+IC50γ)). Where, V is the enzyme activity; V0 is the enzyme activity in the absence of

test compound; V∞ is the remaining enzyme activity when the concentration of test compound is

infinite; C is the concentration of test compound; IC50 is the concentration of test compound that

causes 50% inhibition of V0; and γ is the sigmoidicity factor.

NADPH Dependence and Irreversibility of P450 Enzyme Activity Inhibition. The

experiments described in this section were conducted for the P450 enzymes in which TDI was

observed: CYP1A2, 2B6 and 3A4/5 (testosterone and midazolam). The HLM incubations were


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conducted at a single concentration of irosustat (5 µM for CYP1A2 and 50 µM for CYP2B6 and

CYP3A4/5), 667-coumarin (0.5 µM for CYP1A2 and 5 µM for CYP2B6 and CYP3A4/5), and

reference mechanism-based inhibitors (2 µM furafylline, 4 µM thio-TEPA, and 50 µM

mifepristone, respectively). Appropriate controls without test compound or reference inhibitor

were included. Incubations were performed at 37ºC as indicated in Table 1 in triplicate. For

CYP3A4/5 assays the following parameters varied: 10 minutes incubation time, 0.5 ml

incubation volume and 0.1 or 0.15 mg/ml protein concentration for midazolam or testosterone

substrates, respectively. To evaluate NADPH dependence of P450 activity inhibition, HLM were

preincubated at 37°C with irosustat, 667-coumarin, or reference inhibitor for 0, 15, and 30 min in

the presence or absence of NADPH generating system before addition of each P450 substrate.

The irreversibility of P450 activity inhibition was assessed also by preincubation of HLM under

the same conditions but with a 25-fold increase in microsomal protein concentration (7.5 mg/ml

for CYP1A2 and CYP2B6, 2.5 mg/ml for CYP3A4/5-midazolam and 3.75 mg/ml for CYP3A4/5-

testosterone). After preincubation, the mixtures were diluted 1/25 prior addition of each P450

substrate.

Determination of Ki . Ki parameter was determined for CYP1A2 and CYP2C19 activity

inhibition by 667-coumarin. Incubations with HLM were performed using five concentrations of

each specific substrate (from 20 to 240 µM phenacetin; or from 10 to 200 µM S-mephenytoin)

and five concentrations of 667-coumarin (from 0 to 5 µM for CYP1A2; or from 0 to 25 µM for

CYP2C19). All incubations were performed at 37ºC in triplicate, following specific incubation

conditions for each P450 assay (Table 1) without preincubation step. The following equations

(Cheng and Prusoff, 1973) were fitted to the individual enzyme activity data by means of

simultaneous non-linear regression using WinNonlin software.


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Competitive inhibition: V=((Vmax·[S])/(KM·(1+([I]/Ki))+[S]))

Non-competitive inhibition: V=((Vmax·[S])/(KM·(1+([I]/Ki))+[S]·(1+([I]/Ki))))

Uncompetitive inhibition: V=((Vmax·[S])/(KM+[S]·(1+[I]/Ki)))

where V and Vmax are the observed and maximal enzymatic activity, [S] is the substrate

concentration, [I] is the 667-coumarin concentration, KM is the Michaelis-Menten constant and Ki

is the inhibition constant. The Akaike Criteria and Schwarz´s Bayesian Criteria, together with the

coefficient of variation of the Ki estimates, were considered to select the inhibition model. The

lowest values of these parameters indicated the model that best fitted to the data.

UGT Inhibition. The potential inhibitory effect of irosustat and 667-coumarin on the

main UGTs involved in drug metabolism: UGTs 1A1, 1A4 and 2B7 (Williams et al., 2004) was

investigated as follows:

Inhibition Assays for UGT1A1 and UGT2B7 Activities. Prior to the inhibition

experiments, the linearity of 7-hydroxy-4-methylcoumarin glucuronosyltransferase activity by

recombinant human UGT1A1 and UGT2B7 was evaluated as a function of protein concentration

and incubation time. Linear metabolite formation was found up to 0.5 and 1 mg protein/ml for

UGT1A1 and UGT2B7, respectively, and up to 60 minutes for both UGTs (data not shown).

These incubation conditions were selected for the following experiments. Recombinant UGTs

1A1 and 2B7 were incubated separately in triplicate with 7-hydroxy-4-methylcoumarin as

substrate (working solutions in dimethylsulfoxide (DMSO), respectively) at a concentration

similar to the KM for each enzyme [113 and 335 µM, respectively (Uchaipichat et al., 2004)], in

the presence of 0 to 50 µM irosustat or 667-coumarin. The incubations were performed at 37ºC in


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100 mM sodium phosphate buffer pH 7.4 containing 10 mM MgCl2, 2 mM UDP-glucuronic acid

and 0.025 mg/ml alamethicin (0.4 ml final volume). The reactions were started by addition of

UDP-glucuronic acid and were quenched by addition of acetonitrile containing 7-

hydroxycoumarin as internal standard. 50 µM diclofenac was used as reference inhibitor

(Uchaipichat et al., 2004).

Inhibition Assay for UGT1A4 Activity. Linear conditions for trifluoperazine

glucuronosyltransferase activity by UGT1A4 were described in the certificate of analysis of the

recombinant enzyme. Recombinant human UGT1A4 (0.4 mg/ml) was incubated in triplicate with

50 µM trifluoperazine (close to its KM, Zhang et al., 2005, working solutions prepared in

DMSO), in the presence of 0 to 50 µM irosustat or 667-coumarin. The incubations were

performed as described for UGTs 1A1 and 2B7 but using 50 mM Tris·HCl pH 7.4 as incubation

buffer. The reactions were quenched after 30 minutes by addition of one volume of acetonitrile

containing 6% acetic acid. 1.6 mM quinidine was used as reference inhibitor (Uchaipichat et al.,

2006).

Incubation mixtures without substrate, without UDP-glucuronic acid, and without UGT

were used as controls (n=1).

P450 Enzyme Induction. 0.25, 2.5 and 10 µM irosustat (n=4), was incubated at 37°C for

72 hours (CYP1A2 and CYP3A4 assays) or 96 hours (CYP2C9 and CYP2C19 assays) with

freshly isolated human hepatocytes from four individual women donors (one of the batches -

batch HEP200116- failed to provide valid data for induction of CYP2C9). Prototypical P450

inducers were incubated (n=4) as positive controls at 50 µM concentration: omeprazole for

CYP1A2 and rifampicin for CYP2C9, 2C19 and 3A4 (both dissolved in DMSO). Incubations


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without any compound but the same organic solvent composition (0.2% acetonitrile or DMSO)

were also conducted as negative controls (n=4). Hepatocytes were used as monolayer cultures in

96-well plates coated with rat type I collagen. They were seeded at a density of 50,000 cells/well,

and were allowed to attach at 37°C in 0.1 ml culture medium [Williams’E + Glutamax I medium

(Invitrogen, Carlsbad, CA) supplemented with 10% foetal calf serum, 100 IU/ml penicillin, 100

µg/ml streptomycin, and 4 µg/ml bovine insulin] in an humidified chamber with air containing

5% CO2. After cell attachment, the culture medium was replaced by the incubation medium

(Williams’E + Glutamax I medium supplemented with 50 µM hydrocortisone, 100 IU/ml

penicillin, 100 µg/ml streptomycin, and 4 µg/ml bovine insulin) which contained irosustat or the

prototypical inducers. Because irosustat is unstable in the incubation medium at 37ºC and pH 7.4,

the medium was renewed every 12 hours (also for positive and negative controls). Prior to and

during incubations, cell morphology was evaluated by optic microscopy in order to detect any

sign of cytotoxicity. At the end of the incubation period, cytotoxicity was also assessed by the

neutral red uptake test.

Incubation Conditions to Determine P450 Activities. At the end of the treatment periods,

incubation media were removed and cells were washed with 25 mM HEPES buffer pH 7.4

(Sigma-Aldrich). Cells were then incubated with MEM Eagle medium devoid of phenol red

(VWR International, Radnor, PA) in the presence of probe substrates for CYP1A2, 2C9, 2C19

and 3A4 (200 µM phenacetin, 1 mM tolbutamide, 200 µM S-mephenytoin and 200 µM

nifedipine, respectively). At the end of each incubation period (2 h for CYP3A4/5 assay, and 6 h

for the remaining assays), the supernatants and the cell monolayers were stored separately at -

80°C. Metabolite formation from P450-probe substrates was determined in the supernatants by

HPLC (see analytical methods section, Table 2, and supplemental data), while cells were lysed by


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heating at 37°C for 15 to 60 minutes in 100 µL 0.01N NaOH. Protein content in cell lysates was

determined using DC Protein Assay Kit (Bio-Rad, Hercules, CA), and was used for the enzyme

activity calculation. P450 activities were expressed as nanomoles of metabolite formed per hour

and per mg of cell proteins. Induction was calculated as the fold change of enzyme activity in

each batch of hepatocytes, and for each treatment group relative to vehicle control.

Incubation Conditions to Measure P450 mRNAs. In a separate experiment, 2.5 µM

irosustat or the prototypical inducers (see above) were incubated in triplicate with two individual

batches of female human hepatocytes for CYP1A2, CYP2C19 and CYP3A4 mRNA

quantification. Hepatocyte monolayers were prepared in 24-well plates coated with rat type I

collagen, and were seeded at a density of 380,000 cells/well (0.5 ml culture medium) following

the same conditions described above. The duration of the incubations was 48 hours. In parallel,

hepatocytes from both batches were incubated with 2.5 µM irosustat during 96 hours for

CYP2C19 enzyme activity evaluation. In both cases the medium was renewed every 12 hours.

Expression of mRNA was determined using SYBR Green-based real time quantitative

polymerase chain reaction (Real Time PCR) after RNA extraction and reverse transcription (RT).

Effect of AIs on the In Vitro Metabolism of Irosustat. Pooled HLM (1 mg protein/ml)

were incubated at 37°C in triplicate with 50 mM Tris·HCl buffer pH 7.4 containing 50 µM

irosustat for 40 minutes, in the presence and absence of increasing concentrations of each AI

(letrozole, anastrozole and exemestane) and NADPH-generating system. The incubation time and

microsomal protein concentration were selected to be in the linear range of irosustat metabolism

in HLM, while irosustat concentration was set to be close to its KM value in HLM (Ventura et al.,

2011). Two incubation conditions were used: (a) simultaneous incubation of irosustat with the

AI; or (b) 30 minutes preincubation of HLM with the AI in the presence of cofactor before the


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addition of irosustat. AI concentrations ranged from 0 to 100 µM for letrozole and anastrozole,

and from 0 to 20 µM for exemestane. Incubations were quenched with one volume of 10% acetic

acid in acetonitrile and samples were analysed by HPLC-UV to evaluate the irosustat metabolite

formation as previously described (Ventura et al., 2011). The following solvents were used to

dissolve irosustat (acetonitrile), letrozole and anastrozole (DMSO), and exemestane (methanol).

The solvent composition was adjusted to be constant in all assays and did not exceed 2% of the

total incubation volume). Specific reference inhibitors of the main P450s involved in irosustat

phase I metabolism were used at a single active concentration to validate the experiments: 10 µM

quercetin, 2 µM sulfaphenazole and 0.5 µM ketoconazole (CYP2C8, 2C9 and 3A4/5,

respectively). Incubation mixtures without irosustat and AI, without HLM, and without β-NADP

were used as controls (n=1). The peak area values of each irosustat metabolite were transformed

into percentage of inhibition as compared to control samples without AI.

Analytical Methods.

Methodologies for P450 Enzyme Activity Determination. After incubation, the reactions

were quenched by the addition of organic solvent and/or in acidic conditions plus the internal

standard (if used, see Table 2). For CYP2C9, samples were extracted with diethyl ether. Analysis

were conducted by HPLC using an Alliance 2695 module or a 600 solvent delivery system

(Waters, Milford, MA). For CYP2B6 and CYP2C19, on-line solid phase extraction was

performed automatically (PROSPEKT system, Spark Holland). The UV detection of the

metabolites was performed using 2487, 2489 or 486 detectors (Waters), except for CYP2D6

where fluorescence detection was used (2475 detector, Waters). Analytical conditions are

described in Table 2. Supplemental data describes specific analytical conditions used for

CYP1A2 and CYP2C19 assays in human hepatocytes during induction experiments, and


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CYP3A4/5 assays in HLM during the assessment of NADPH dependence and the irreversibility

of P450 inhibition).

Methodology for 7-Hydroxy-4-methylcoumarin Glucuronosyltransferase Enzyme Activity

Determination. The incubates were centrifuged at 16000 x g at 4°C for 15 min, the supernatants

were diluted with mobile phase and analysed by HPLC using an Alliance 2695 module (Waters)

equipped with a Symmetry C18 column (150 x 4.6 mm, 5 µm particle diameter, Waters) at room

temperature. The mobile phases were 50 mM ammonium acetate pH 5.0 (solvent A) and

acetonitrile (solvent B), which were flushed at 1 ml/min. The initial mobile phase contained 8%

solvent B which was increased linearly up to 50% over the next 10 min, and further increased to

90% in 1 min. The percentage of solvent B was maintained at 90% for 3 min, and was set to the

initial conditions for the last 8 min. The UV detection of the metabolite, 7-hydroxy-4-

methylcoumarin glucuronide, was performed with a 2487 detector (Waters) operating at 325 nm.

Methodology for Trifluoperazine Glucuronosyltransferase Enzyme Activity

Determination. The incubates were centrifuged and the supernatants diluted with mobile phase as

described. Sample analysis were conducted using the same HPLC system, detector and column as

for 7-hydroxy-4-methylcoumarin glucuronosyltransferase assay, but operating at 45ºC. The

mobile phases were 0.1% TFA (solvent A) and 0.1% TFA in acetonitrile (solvent B), which were

flushed at 1 ml/min. The initial mobile phase contained 30% solvent B which was maintained for

1.2 min and further increased linearly up to 51% over the next 8.4 min. Finally, the percentage of

solvent B was set to the initial conditions for the last 5 min. The UV detection of the metabolite,

trifluoperazine glucuronide, was performed at 256 nm.


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RNA extraction. At the end of the exposure periods, hepatocytes were washed with PBS

and further kept at -80°C. Total cell RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden,

Germany). The RNA content in each extract was quantified by UV absorption at 260 nm. The

ratio between the optical density at 260 nm and 280 nm was used to evaluate the purity of the

specimens. The integrity of RNA in each extract was checked by electrophoresis on agarose gel.

Reverse transcription (RT). The mRNA was converted to cDNA using the reverse

transcriptase enzyme (Super Script II RT, Invitrogen). RT was performed by heating the

specimens at 42°C for 30 min in the presence of deoxyribonuclotides triphosphate, random

hexamers 5’-phosphate, dithiothreitol, and recombinant RNasin ribonuclease inhibitor (Promega,

Madison, WI), The same amount of RNA was used for all specimens.

Real Time PCR. It was performed on a MiniOpticon Real Time PCR system (Bio-Rad)

using SYBR Green I (Applied Biosystems, Carlsbad, CA) and a validated set of primers of

CYP2C19, CYP1A2 and CYP3A4 (PrimSign primers, Biopredic International). The following

housekeeping genes, TBP (coding for the TATA-box binding protein), RPLP0 (P0; coding for

large ribosomal protein) and Cyclophiline A, were analysed as the reference genes (Girault et al.,

2005). cDNA was amplified by PCR in the presence of SYBR Green I, Taq polymerase,

deoxyribonuclotides triphosphate, MgCl2 and specific primers of the screened gene. In all PCR,

amplification was conducted in duplicate on diluted RT products. Each PCR assay included 40

cycles. For one cycle the program was as follows: 15 seconds at +95°C for DNA denaturation, 1

minute at +65°C for amplification and reading of fluorescence. At the end of the PCR, double

strains of DNA were dissociated by progressive increase of temperature to obtain the melting

curve for each set of primers to control specific amplification. The mean of the duplicate

threshold cycle (CT) values for each specimen was determined and the CT of each P450 was


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normalized by each reference gene (i.e. ΔCT calculation). Results, expressed as fold induction,

were determined by the formula 2ΔCT of the treated samples normalized by control samples

treated (vehicle).

Statistical Analysis. The following statistical tests for the CYP2C19 inhibition observed

during the P450 induction experiments were performed using SigmaStat software version 1.0

(Jandel Scientific, Germany): one-way analysis of variance followed by a Bonferonni’s test, or a

t-test (α<0.05). These tests were carried out for each hepatocyte batch to determine if there were

statistically significant differences between the group means.


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Results

P450 Enzyme Activity Inhibition. The inhibitory potential of irosustat on CYP1A2,

2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4/5 (CYP3A4/5 substrates: nifedipine, midazolam

and testosterone) was studied in HLM comparing the P450-specific activities at increasing

concentrations of test compound, with and without 30 minute preincubation in the presence of the

irosustat and cofactor to assess possible TDI. As shown in Table 3, the results let us to classify

P450s in three categories as a function of the extent of enzyme activity inhibition by irosustat: (a)

non-inhibited P450 enzyme activities: CYP2A6, 2C8, 2E1 and 3A4/5 (nifedipine substrate),

which were not inhibited at any of the assayed concentrations; (b) mildly inhibited P450 enzyme

activities: CYP2B6, 2C9, 2D6 and 3A4/5 (midazolam and testosterone substrates), which were

inhibited only at the highest tested irosustat concentrations and resulted in an estimated IC50 value

higher or similar to 50 µM; and (c) highly inhibited P450 enzyme activities: CYP1A2 and to a

lesser extent CYP2C19, for which IC50 values were estimated to be below 50 µM (the estimated

IC50 parameters are presented in Table 3). For CYP1A2, 2B6 and 3A4 (with midazolam and

testosterone as substrates), TDI was observed. All the used reference P450-inhibitors (see Table

1) showed the expected inhibitory effect validating the experiments.

The potential inhibitory effect of the irosustat derivative 667-coumarin on CYP1A2 and

CYP2C19 was also investigated. The IC50 parameters presented in Table 4 show that 667-

coumarin was more potent than irosustat in inhibiting both P450 enzyme activities. For CYP1A2,

667-coumarin showed an IC50 value more than ten-fold lower than for the parent compound

without preincubation (0.65 µM vs 7.6 µM), and four-fold lower after 30 minute preincubation

with HLM and cofactor (0.27 µM vs 1.1 µM). The remarkable differences between both


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incubation conditions demonstrated TDI for both compounds on CYP1A2 activity. For

CYP2C19, 667-coumarin showed an IC50 value three- to four-fold lower than for irosustat.

However, the potency of irosustat and 667-coumarin on the inhibition of CYP2C19 activity was

remarkably lower than for CYP1A2, and preincubation did not enhance 667-coumarin-induced

inhibition of CYP2C19 activity.

Since 667-coumarin was more potent than irosustat in inhibiting both CYP1A2 and

CYP2C19 activities, additional incubations were performed in order to determine the Ki

parameter and the inhibition type of 667-coumarin on both P450 enzyme activities. The resulting

Ki values are shown in Table 4. As expected, 667-coumarin was more potent in inhibiting

CYP1A2 (Ki = 0.77 µM) than CYP2C19 (Ki = 5.8 µM) and the competitive inhibition model

was the one that statistically best fitted to the inhibition data of 667-coumarin on both enzymes.

As TDI was found for CYP1A2, 2B6 and 3A4/5 (using midazolam and testosterone as

substrates), the effect of NADPH-dependence and the irreversibility of the inhibition of these

P450 activities were assayed for both irosustat and 667-coumarin (Table 5). TDI for all the

assayed P450 enzymes was confirmed for irosustat, although the inhibition was not dependent on

NADPH in any of the cases. When the effect of 667-coumarin was assessed, no differences were

found among the preincubation times for CYP2B6 and CYP3A4/5, either in the presence or

absence of NADPH. However, TDI of CYP1A2 activity was caused by 667-coumarin only when

NADPH was present. The results of the dilution experiments showed that the CYP1A2, 2B6 and

3A4/5 inhibition by irosustat and 667-coumarin can be considered as reversible. The reference

mechanism-based P450 inhibitors (2 µM furafillyne for CYP1A2, 4 µM thio-THEPA for

CYIP2B6 and 50 µM mifepristone for CYP3A4/5) were able to produce time-dependent,


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NADPH-dependent and irreversible inhibition of their corresponding P450s validating the

experimental procedure (Table 5).

UGT Enzyme Activity Inhibition. The inhibitory potential of irosustat and 667-

coumarin towards UGT1A1, UGT1A4 and UGT2B7 was studied and the estimated IC50

parameters are presented in Table 6. Both, irosustat and 667-coumarin were able to inhibit almost

all UGT enzyme activities at the highest tested concentration (50 µM), except for irosustat on

UGT1A4 activity for which no inhibition was observed at the working concentration range. In all

assays, 667-coumarin showed always higher inhibitory potential than irosustat. UGT1A1 activity

was inhibited at the highest extent by both compounds (IC50 values were 43 µM and 30 µM for

irosustat and 667-coumarin, respectively).

P450 Enzyme Induction. Irosustat at 0.25, 2.5 and 10 µM was incubated with freshly

isolated human hepatocytes for 72 hours (to assess CYP1A2 and CYP3A4 induction) or 96 hours

(to assess CYP2C9 and CYP2C19 induction). Hepatocytes corresponded to three separate

donors. Cell viability after isolation was determined by the trypan blue exclusion method and

ranged from 75% to 94%. The prototypical P450 inducers (50 µM omeprazole for CYP1A2 and

50 µM rifampicin for CYPs 2C9, 2C19 and 3A4) showed the expected results (≥2-fold

induction), validating the induction response of the three hepatocyte batches. Results from each

individual donor are shown in Figure 2.

CYP1A2 Enzyme Activity. Phenacetin O-deethylase activity was not appreciably changed

at any of the tested irosustat concentrations in two of the hepatocyte batches. In batch

HEP200124, CYP1A2 enzyme activity was increased by a factor 1.6 and 1.4 relative to solvent


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control, at 2.5 and 10 µM irosustat, respectively, but representing only 12.3% and 10.8% of the

positive control omeprazole.

CYP2C9 Enzyme Activity. Tolbutamide methylhydroxylase activity was not noticeably

changed at any of the tested concentrations of irosustat in any of the batches of hepatocytes used.

CYP2C19 Enzyme Activity. In the presence of irosustat, S-mephenytoin 4’hydroxylase

activity significantly decreased to 40-70% of the control value whatever the tested concentration

of test compound (even at the lowest concentration 0.25 µM) and for the three hepatocyte

batches.

CYP3A4/5 Enzyme Activity. Nifedipine oxidase activity was not appreciably changed at

any of the tested irosustat concentrations in one of the hepatocyte batches. In batch HEP200121,

CYP3A4/5 enzyme activity was increased by a factor 1.2 and 1.3 relative to solvent control after

exposure to 2.5 µM and 10 µM irosustat, respectively. On the other hand, in batch HEP200124,

CYP3A4/5 enzyme activity decreased to 70% of control value after treatment with 10 µM

irosustat.

Measurement of mRNA Expression. In a second induction experiment, 2.5 µM irosustat

was incubated with human hepatocytes from two individual donors for 48 hours to evaluate

CYP1A2, CYP2C19 and CYP3A4 mRNA content, and for 96 hours to assess CYP2C19 enzyme

activity. Cell viability after isolation was determined by the trypan blue exclusion method and

ranged from 96% to 98%. The prototypical inducers used to validate the PCR results (see above),

worked as expected. Results are shown in Figure 3. As occurred in the preceding experiments,

CYP2C19 enzymatic activity significantly decreased to 50%-60% of control after exposure to 2.5

µM irosustat. The expression of CYP2C19, CYP1A2 and CYP3A4 mRNA was evaluated on the


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same hepatocytes. No considerable modification of CYP2C19 and CYP3A4 mRNA expression in

cells exposed to irosustat for 48 hours was detected in the two batches of hepatocytes (results

between 0.8-1.2 fold induction relative to solvent control). In contrast, the expression of CYP1A2

mRNA was increased to 2.9 and 2.3-fold of control (acetonitrile 0.2%) in hepatocytes from

batches HEP200169 and HEP200172, respectively. However, this increase represented only 5%

and 1% of the response to the positive inducer omeprazole, respectively.

Effect of AIs on the In Vitro Metabolism of Irosustat. The inhibitory potential of the

AIs letrozole, anastrozole and exemestane on the in vitro metabolism of irosustat was studied

using HLM with and without preincubation of the AIs in the presence of cofactor. The metabolite

profile of irosustat in HLM was characterised by the formation of 10 main phase I metabolites in

addition to 667-coumarin, namely M7, M8, M9, M11, M13, M14, M15, M16, M18 and P-36. For

letrozole, the inhibition effect on the formation of irosustat metabolites never reached 50% at the

concentration range tested (up to 100 µM), particularly formation of metabolites M14 and M15

was completely unaffected. Because of that, the IC50 parameters were not estimated. Similar

results were obtained with exemestane which did not affect the formation of any irosustat

metabolite at the concentration range tested (up to 20 µM). On the contrary, anastrazole (from 5

µM concentration) caused relevant inhibition of irosustat metabolism, either with or without 30

min preincubation in the presence of cofactor. Table 7 shows the estimated IC50 parameters for

anastrozole corresponding to each irosustat metabolite. IC50 values ranged from 38 µM to >100

µM in the condition without preincubation, and from 34 µM to 83 µM after 30 min

preincubation. Comparatively, M13 showed the most inhibited formation rate. The respective

CYP2C8, CYP2C9 and CYP3A4/5 specific enzyme activity inhibitors: quercetin, sulfaphenazole


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and ketoconazole, worked as expected on inhibiting the formation of irosustat metabolites

(Ventura et al., 2011).


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Discussion

To support the clinical development of irosustat, we evaluated in vitro its inhibitory

potential on the main drug metabolizing P450s and UGTs, together with its capability to induce

CYP1A2, 3A4, 2C9 and 2C19. The desulfamoylated irosustat metabolite 667-coumarin was

included in some of the investigations. The effect of the most frequently prescribed AIs in breast

cancer on irosustat metabolism was also studied.

The hepatic P450s whose activity was inhibited most by irosustat were CYP1A2 and

CYP2C19 (Table 3), for which the effect of 667-coumarin was also evaluated (Table 4). 667-

coumarin was a more potent inhibitor of CYP2C19 activity than irosustat. No differences

appeared between conditions with and without preincubation indicating absence of TDI. The

metabolite profile of irosustat was determined in some samples by HPLC showing a decrease in

irosustat concentration following preincubation, while 667-coumarin levels remained nearly

constant (data not shown). This is because 667-coumarin is formed from irosustat in a non

NADPH-dependent process that is balanced by its own P450-mediated metabolism (Ventura et

al., 2011). These data indicate that 667-coumarin is probably causing CYP2C19 inhibition in

HLM. In fact, 667-coumarin is a competitive inhibitor of CYP2C19 showing a Ki of 5.8 µM

(Table 4), a concentration approximately 40-fold the highest 667-coumarin plasma Cmax,ss in

humans following irosustat recommended dose. Surprisingly, when irosustat was incubated with

human hepatocytes, a significant non dose-dependent inhibition of CYP2C19 enzyme activity

was observed (30 to 60% inhibition at irosustat concentrations as low as 0.25 µM, see Figure 2).

Therefore, results in HLM are not sufficient to explain the remarkable effect on CYP2C19 in

hepatocytes, even considering that all irosustat is transformed to 667-coumarin. Furthermore,


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CYP2C19 mRNA content in hepatocytes was unaffected by exposure to irosustat, indicating no

repression of CYP2C19 gene. Since the metabolite profile of irosustat in hepatocytes is markedly

different from the profile in HLM, basically consisting in conjugations with glucuronic acid and

sulfate (Ventura et al., 2011), an inhibitory effect of any irosustat phase II metabolite might

explain the strong inhibition of CYP2C19 activity. Moreover, since hepatocytes are a more

complex test system than microsomes, other possible mechanisms (i.e. involvement of hepatic

drug transporters leading to increased intracellular concentrations of test compounds or

metabolites) may not be excluded. Further studies are needed to investigate the mechanism

underlying CYP2C19 inhibition by irosustat or any of its metabolites.

The inhibition of CYP1A2 activity by irosustat and 667-coumarin in HLM was

remarkably higher than for CYP2C19 (Tables 3 and 4). Again, 667-coumarin showed higher

inhibition potential than its parent compound. In this case clear differences between the two

incubation conditions were found that required additional experiments. As shown in Table 5,

inhibition of CYP1A2 activity by irosustat increased with the preincubation time in a NADPH-

independent manner suggesting a role of 667-coumarin on CYP1A2 inhibition, because only

667-coumarin may be formed from irosustat in the absence of NADPH. This was further

demonstrated using HLM incubated with 667-coumarin which caused 40% decrease of CYP1A2

activity in the absence of NADPH regardless of the preincubation time. However, inhibition of

CYP1A2 activity increased by 30% after 30 min preincubation with NADPH, and was reversed

by dilution. Therefore, we concluded that CYP1A2 inhibition is most probably mediated by 667-

coumarin and by its metabolites acting as reversible inhibitors (Ogilvie et al., 2008; Grimm et al.,

2009). 667-coumarin is a competitive CYP1A2 inhibitor showing a Ki value of 0.77 μM, (Table

4), a concentration exceeding only by 5-fold its highest plasma Cmax,ss in humans following


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irosustat recommended dose. These data indicate that DDI is possible in the clinical setting as a

result of CYP1A2 inhibition in case of co-medication of CYP1A2 substrates with irosustat (FDA

draft guidance, 2006). Moreover, the lower IC50 values for 667-coumarin when preincubated with

NADPH (Table 4) strongly suggest that some 667-coumarin phase I metabolites are even more

potent CYP1A2 inhibitors. These results point out the need of clinical evaluation of DDI on

CYP1A2.

CYP2B6, 2C9, 2D6 and 3A4/5 activities were inhibited by irosustat only at the highest

assayed concentration (50 μM, see Table 3). This inhibition is not clinically relevant because 50

µM irosustat represents approximately 120-fold the plasma Cmax,ss in humans. As shown in Table

5, CYP2B6 and CYP3A4/5 activities were inhibited in a time-dependent manner, but the

presence of NADPH did not enhance inhibition, meaning that no mechanism-based inhibition

was produced. Besides, 667-coumarin is not likely to inhibit these enzymes in vivo in humans.

The remaining P450s: CYP2A6, 2C8, 2E1 and 3A4/5 (nifedipine as substrate) were unaffected

by irosustat.

From UGT inhibition data (Table 6), neither irosustat nor 667-coumarin are likely to

affect the activity of the main drug metabolizing UGTs (Williams et al., 2004). UGT1A1 was the

highest inhibited enzyme but the IC50 values for irosustat and 667-coumarin for the inhibition of

this enzyme are far from their clinically relevant plasma concentrations. The higher inhibitory

potential of 667-coumarin towards UGT1A1 and UGT2B7 activities as compared to irosustat

may be explained by the fact that 667-coumarin is directly metabolized by these enzymes to its

glucuronide (M12, Ventura et al., 2011).


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Irosustat up to 10 µM did not induce CYP1A2, 2C9, 2C19 and 3A4/5 following the

criteria described in the current guidelines (FDA draft guidance, 2006: positive induction when

enzyme activity or mRNA is >40% of positive control; EMA draft guidance 2010: positive

induction when enzyme activity is >50%, or mRNA is >20% of positive control, respectively).

None of the results showed an increase in enzyme activity higher than 1.5-fold, except for

hepatocyte batch HEP200124 exposed to 2.5 µM irosustat, which gave 1.6-fold higher CYP1A2

activity but only represented 12.3% of the response to the positive control omeprazole. The

inhibition of CYP2C19 activity found in human hepatocytes has been discussed above.

Because AIs are expected to be potentially used in combination therapy with irosustat for

breast cancer, the prediction of possible DDIs between AIs and irosustat becomes a key factor on

irosustat development. The three mainly prescribed AIs: letrozole, anastrozole and exemestane

were screened. From literature, the three AIs are metabolised mainly by CYP3A4 (Femara®

prescribing information, 2009; Scripture and Figg, 2006; Aromasin® prescribing information,

2005). Besides, CYP2A6 seems to contribute to letrozole biotransformation. Also, a role of

CYP1A enzymes has been attributed to exemestane metabolism together with CYP4A11 and

aldoketoreductases, in a process where compensatory contribution of the various enzymes may

occur (Kamdem et al., 2011). Data from the present work demonstrated neither inhibition of

CYP3A4/CYP2A6 activity nor induction of CYP3A4 by irosustat at clinically relevant

concentrations. However, because AIs could theoretically inhibit P450s involved in irosustat

metabolism, their effect was studied in incubations of irosustat with HLM (Table 7). Letrozole

showed almost no effect on irosustat metabolism (IC50 were higher than 100 µM for all irosustat

metabolites), and exemestane did not inhibit irosustat metabolism up to 20 μM. In both cases,

these concentrations exceed by 200-fold the respective plasma Cmax,ss in humans (Pfister et al.,


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2001; Groenewoud et al., 2010). Anastrozole was slightly more potent than the other two AIs in

inhibiting irosustat metabolism, showing IC50 values between 34 and >100 µM depending on the

irosustat metabolite (Table 7). Therefore the Ki value for the inhibition of irosustat metabolism

by anastrozole should be approximately one half of the IC50 when the substrate concentration in

the experiments is close to its KM (Cheng and Prusoff, 1973), as in the present work. Hence,

making a conservative estimation, our obtained IC50 data indicate that the Ki would be not lower

than 10-20 µM, a concentration 30-fold higher the plasma Cmax,ss of anastrozole following a

standard daily dose of 1 mg (Grimm and Dyroff, 1997). Altogether these results indicate that no

changes in irosustat pharmacokinetics are anticipated as a result of metabolism inhibition in case

of co-medication with AIs.

Summarizing, the data presented in this work strongly suggest that irosustat is not likely

to inhibit or induce most of the P450 enzymes involved in drug metabolism in vivo, except for

CYP1A2 and CYP2C19 inhibition. Likewise, irosustat did not show any inhibitory effect on the

activity of UGT1A1, 1A4 and 2B7 enzymes. The irosustat derivative 667-coumarin notably

inhibited CYP1A2 activity in HLM at clinically relevant concentrations. The inhibition was

reversible and probably enhanced by 667-coumarin metabolites. Clinical interaction studies on

CYP1A2 are recommended. Irosustat inhibited CYP2C19 activity in HLM in a lesser extent

through the formation of 667-coumarin. Since CYP2C19 inhibition increased largely when

assessed in human hepatocytes and CYP2C19 gene expression was unaffected, additional

mechanistic and/or clinical follow-up studies are needed to explain CYP2C19 inhibition by

irosustat. Finally, because the AIs letrozole, anastrozole and exemestane are potential candidates

for irosustat combination therapy, prediction of possible DDIs between AIs and irosustat was

studied. From published data, no effect of irosustat on the pharmacokinetics of these drugs is


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anticipated. The results of the present work demonstrate that the mentioned AIs are not able to

inhibit the formation of the primary irosustat metabolites at clinically relevant concentrations.

Consequently, no change in irosustat pharmacokinetics is expected to occur in vivo.


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Acknowledgements: The authors would like to thank M.C. Gómez, C. Maté and M. Víctor for

their technical assistance.


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Authorship Contributions

Participated in research design: Ventura, Solà and Brée

Conducted experiments: Ventura

Contributed new reagents or analytic tools: Ventura, Solà and Brée

Performed data analysis: Ventura

Wrote or contributed to the writing of the manuscript: Ventura, Solà, Brée, Peraire, and Obach


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Footnotes

This work was sponsored by Ipsen Group.

Reprint requests to be addressed to: Josep Solà

Ipsen Pharma S.A.

Crta. Laureà Miró, 395

08980 Sant Feliu del Llobregat, Barcelona (Spain)

Email: [email protected]


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Legends for Figures

Figure 1. Scheme of irosustat metabolism with chemical structures of irosiustat and 667-

Coumarin.

Figure 2. Induction potential of irosustat on CYP1A2, CYP2C9, CYP2C19 and CYP3A4/5

enzymatic activities in human hepatocytes. First experiment. Results are expressed as fold

induction, mean ± SD, n=4, three donors. Reference inducer: omeprazole for CYP1A2 and

rifampicin for CYPs 2C9, 2C19 and 3A4. Comparison to vehicle control group: *p<0.05,

**p<0.01, and ***p<0.001. Abbreviation: ACN: acetonitrile.

Figure 3. Induction potential of irosustat on CYP2C19 enzymatic activity and CYP1A2,

CYP2C19 and CYP3A4 mRNA expression in human hepatocytes. Second confirmative

experiment. Results are expressed as fold induction, mean ± SD, n=3, two donors. Reference

inducer: omeprazole for CYP1A2 and rifampicin for CYPs 2C9, 2C19 and 3A4. Comparison to

vehicle control group: ***p<0.001. Abbreviation: ACN: acetonitrile.


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Table 1. Incubation conditions for P450 inhibition assays in HLM. 100 mM sodium

phosphate buffer pH 7.4 was used as incubation buffer in all P450 assays except for CYP2A6

assay (50 mM Tris-HCl buffer pH 7.4).

P450

Enzyme Marker Substrate

Protein

Conc.

(mg/ml)

Incub.

Time

(min)

Incub.

Volume

(ml)

Reference Inhibitor

CYP1A2 60 µM phenacetin1 0.3 25 0.5 2 µM furafylline2

CYP2A6 3 µM coumarin2 1 10 0.5 1 µM methoxsalen2

CYP2B6 50 µM bupropion2 0.3 30 1 4 µM thio-tepa2

CYP2C8* 20 µM paclitaxel2 1 20 0.3 30 µM quercetin2

CYP2C9* 200 µM tolbutamide2 0.8 20 0.2 5 µM sulfaphenazole2

CYP2C19 30 µM S-mephenytoin1 0.3 40 1 5 µM NBN†2

CYP2D6 7.5 µM dextromethorphan1 0.3 10 0.5 2 µM quinidine2

CYP2E1* 200 µM chlorzoxazone3 0.5 15 0.3 200 µM disulfiram3

CYP3A4/5* 20 µM nifedipine2 1 5 0.05 0.5 µM ketoconazole2

CYP3A4/5* 10 µM midazolam2 0.3 8 0.25 0.5 µM ketoconazole2

CYP3A4/5* 50 µM testosterone2 0.5 15 0.25 0.5 µM ketoconazole2

* : Incubations were started with 2 mM reduced NADPH, for the remaining assays a NADPH-

generating system was used.

Solvents used: (1) acetonitrile; (2) DMSO; (3) methanol

†NBN : (+)-N-3-benzyl-nirvanol


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Table 2. Analytical methods for the measurement of P450 inhibition in HLM.

P450 Enzyme Metabolite Internal Standard Column Mobile Phases A/B Flow

(ml/min) UV (nm)

CYP1A2 4-acetamidophenol 2-acetamidophenol Kinetex at 50ºC 0.1% ACET_AC / MEOH 0.7 240

CYP2A6 7-OH-coumarin 7-OH-4-methylcoumarin Supelcosil AMMON_ACET / ACN 1.0 325

CYP2B6* OH-bupropion Trazodone Kinetex at 30ºC AMMON_ACET / ACN 0.6 218

CYP2C8 6α-OH-paclitaxel - Nucleosil MEOH/H2O (65/35)† 1.0 230

CYP2C9 OH-tolbutamide Chlorpropamide Nucleosil Na_ACET+25%ACN / ACN 1.0 230

CYP2C19* 4-OH-mephenytoin 5,5-diphenylhydantoin Kinetex at 40ºC 0.1% ACET_AC / ACN 0.8 204 / 240

CYP2D6 Dextrorphan Levallorphan Atlantis at 40ºC AMMON_ACET / ACN 0.7 FL

CYP2E1 6-OH-chlorzoxazone - Nucleosil 0.5% ACET_AC / ACN 1.0 287

CYP3A4/5 Oxidized Nifedipine Nitrendipine Lichrospher TRIS / ACN:ethyl alcohol (50:50) 1.0 240

CYP3A4/5 1’-OH-midazolam Phenacetin Nucleosil KP / ACN:MEOH (375:625) 1.0 220

CYP3A4/5 6β-OH-testosterone - Nucleosil H2O:MEOH:ACN (64:35:1) / (18:80:2) 1.5 254

* : P450 metabolites were determined after on-line solid phase extraction. Cartridges: C-18, 7 µm (CYP2B6) and C-8, EC-SE, 10 µm (CYP2C19)

† : Isocratic conditions. The remaining assays used solvent gradient conditions.

Abbreviations: OH: hydroxy; ACET_AC: acetic acid; MEOH: methanol; AMMON_ACET: 50 mM ammonium acetate pH 5.0; ACN: acetonitrile; Na_ACET: 10

mM sodium acetate pH 4.3; TRIS: 5 mM Tris pH 7.5; KP: 10 mM potassium phosphate; FL: fluorescence detection (235 nm excitation and 310 nm emission).

Columns: Kinetex C18, 100 x 4.6 mm 2.6 µm (Phenomemex); Supelcosil LC-18-DB, 150 x 4.6 mm 5 µm (Supelco); Nucleosil 100-5 C18, 150 x 4.6 mm 5 µm

(Macherey Nagel); Atlantis dC18, 150 x 4.6 mm 5 µm (Waters); and Lichrospher 100RP18, 125 x 4 mm 5 µm (Interchim).

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

DM

D Fast Forw

ard. Published on March 26, 2012 as D

OI: 10.1124/dm

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DMD #44271

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Table 3. P450 enzyme activity inhibition results for irosustat. Determination of IC50.

Results are expressed as µM, and are the mean value from triplicate incubations.

P450 Enzyme Activity

Direct

IC50 (CV%)

TDI

IC50 (CV%)

CYP1A2 Phenacetin O-deethylase 7.6 (22) 1.1 (8)

CYP2A6 Coumarin 7-hydroxylase NA NA

CYP2B6 Bupropion hydroxylase > 50 ~ 50

CYP2C8 Paclitaxel 6α-hydroxylase NA NA

CYP2C9 Tolbutamide methylhydroxylase > 50 > 50

CYP2C19 S-Mephenytoin 4-hydroxylase 40 (18) 38 (13)

CYP2D6 Dextromethorphan O-demethylase > 50 > 50

CYP2E1 Chlorzoxazone 6-hydroxylase NA NA

CYP3A4/5 Nifedipine oxidase NA NA

CYP3A4/5 Midazolam 1-hydroxylase > 50 ~ 50

CYP3A4/5 Testosterone 6β-hydroxylase ~ 50 44 (9)

(CV%): coefficient of variation of the estimates in percentage

TDI IC50: IC50 estimated after 30 min preincubation of irosustat with HLM and NADPH prior to

the addition of substrate.

NA: not assessed. No inhibition found at any of the assayed concentrations (up to 50 µM)


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Table 4. P450 enzyme activity inhibition results for 667-coumarin. Determination of IC50

and Ki. Results are expressed as µM and are the mean value from triplicate incubations.

P450 Enzyme Direct

IC50 (CV%)

TDI

IC50 (CV%) Ki (CV%) Inhibition Type

CYP1A2 0.65 (4) 0.27 (5) 0.77 (7) Competitive

CYP2C19 10 (6) 13 (17) 5.8 (10) Competitive


TDI IC50: estimated IC50 after 30 min preincubation of 667-coumarin with HLM and

NADPH prior to the addition of substrate.


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Table 5. P450 enzyme activity inhibition results for irosustat and 667-coumarin.

Determination of NADPH-dependence and irreversibility. Irreversibility (dilution test): test

compounds were preincubated with HLM with 25-fold increased microsomal protein

concentration and with NADPH. Incubates were diluted 25-fold prior addition of P450 substrate.

During incubation the actual concentrations of irosustat and 667-coumarin were 2 µM irosustat

and 0.2 µM 667-coumarin (CYP2B6, CYP3A4/5); 0.2 µM irosustat and 0.02 µM 667-coumarin

(CYP1A2). Results are expressed as mean percentage of remaining P450 enzyme activity (n=3).

P450

Enzyme Test Compound

Incubation Conditions and Preincubation Time

+ NADPH

- NADPH

+ dilution

0 min

15 min

30 min

0 min

15 min

30 min

0 min

15 min

30 min

CYP1A2

5 µM Irosustat 73 48 31 69 53 43 99 94 94

0.5 µM 667-Coumarin 65 44 37 61 55 58 104 96 95

2 µM Furafylline 53 21 14 56 49 52 99 65 55

CYP2B6

50 µM Irosustat 77 69 62 nd 62 58 102 98 90

5 µM 667-Coumarin 82 77 79 nd 75 76 113 102 91

4 µM Thio-TEPA 70 47 <31 nd 79 74 90 47 39

CYP3A4/51

50 µM Irosustat 86 76 67 nd 76 69 121 101 95

5 µM 667-Coumarin 99 98 88 nd 101 104 111 104 103

50 µM Mifepristone 30 11 10 nd 29 33 67 22 14

CYP3A4/52

50 µM Irosustat 48 40 36 nd 46 38 89 98 86

5 µM 667-Coumarin 74 69 67 nd 80 76 87 107 102

50 µM Mifepristone 15 <11 <11 nd 17 13 73 12 <6

CYP3A4/5 activities: midazolam 1’-hydroxylation1 and testosterone 6β-hydroxylation2

nd: samples not prepared. The same values as +NADPH (0 min) were assumed.


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Table 6. UGT enzyme activity inhibition results for irosustat and 667-coumarin. Determination

of IC50. Results are expressed as µM and are the mean value from triplicate incubations.

UGT Enzyme Activity

Irosustat 667-Coumarin

Direct

IC50 (CV%)

Direct

IC50 (CV%)

UGT1A1 7-Hydroxy-4-methylcoumarin

glucuronosyltransferase 43 (9) 30 (9)

UGT1A4 Trifluoperazine

glucuronosyltransferase NA > 50

UGT2B7 7-Hydroxy-4-methylcoumarin

glucuronosyltransferase > 50 ~ 50


NA: not assessed. No inhibition found at any of the assayed concentrations (up to 50 µM)


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Table 7. Inhibition of irosustat metabolite formation by the aromatase inhibitors letrozole,

anastrozole and exemestane. Determination of IC50. Results are expressed as µM and are the

mean value from triplicate incubations.

Irosustat

Metabolites

Letrozole Anastrozole Exemestane

Direct

IC50

TDI

IC50

Direct

IC50 (CV%)

TDI

IC50 (CV%)

Direct

IC50

TDI

IC50

M7 >100 >100 78 (5) 66 (5) NA NA

M8 >100 NA 55 (5) 44 (5) NA NA

M9 >100 >100 75 (7) 64 (7) NA NA

M11 >100 >100 >100 83 (13) NA NA

M13 >100 >100 38 (4) 34 (4) NA NA

M14 NA NA 80 (4) 58 (4) NA NA

M15 NA NA 78 (9) 48 (10) NA NA

M16 >100 NA 52 (4) 42 (5) NA NA

M18 >100 >100 42 (4) 41 (4) NA NA

P-36 >100 >100 74 (5) 53 (4) NA NA


TDI IC50: IC50 estimated after 30 min preincubation of the aromatase inhibitor with HLM and

NADPH prior to the addition of irosustat.

NA: not assessed. No inhibition found at any of the assayed concentrations (up to 100 µM

letrozole and 20 µM exemestane)


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